Alzheimer's disease (AD) is the most common cause of dementia in the elderly. To meet the public health challenge posed by AD, a goal set by the National Alzheimer's Project Act is to prevent or treat AD by 2025. Therefore currently there is an urgent need for new therapeutic target discovery and corresponding compound development. A protein deposited in AD brains called amyloid-beta (Abeta) has been hypothesized to play a critical role in AD pathogenesis. Abeta can activate microglia to clear Abeta but at the same time also stimulate microglia to release cytotoxic substances that cause neuronal damage. Using the small molecule TRAM-34, which was synthesized by our group, as a pharmacological tool we recently demonstrated that the calcium-activated potassium channel KCa3.1 plays an important role in microglia activation and microglial neurotoxicity. In vivo evidence indicates that blockade of KCa3.1 by TRAM-34 can inhibit microglia-mediated neuronal killing without affecting their migration and phagocytotic activities. Relevant to AD, our results suggest that TRAM-34 blocks the neurotoxicity induced by Abeta-activated microglia, but does not inhibit their beneficial function of phagocytosing Abeta. KCa3.1 blockade, therefore, is a potential new approach for the treatment of AD. With the help of this grant we wish to perform proof-of-principle studies to validate KCa3.1 as a novel therapeutic target for reducing microglia-mediated neurotoxicity and microglial dysfunction in Alzheimer's disease (AD), through the following three Specific Aims: Aim-1: Determine the effect of KCa3.1 blockade on Ab-induced microglial activation. We will treat cultured microglia with Abeta aggregates (oligomer and fibril) and evaluate the effect of KCa3.1 blockade by TRAM-34 treatment (Aim-1a) or genetic knockout (Aim-1b) on chemotactic and phagocytotic activities, signaling pathways, and the production of chemokines, cytokines, reactive oxygen species, and nitric oxide. Aim-2: Assess the contribution of KCa3.1 to AD-like pathology and cognitive deficits seen in the APPswe/PS1dE9 (APP-PS1) model using the genetic knockout approach. We will cross-breed APP-PS1 mice with KCa3.1-/- mice and evaluate whether KCa3.1 reduction affects neuropathological and behavioral abnormalities in APP-PS1 mice. We will further determine if KCa3.1 reduction can alleviate microglial dysfunction seen in APP-PS1 mice. Aim-3: Validate KCa3.1 as a therapeutic target for AD by performing preclinical studies with TRAM-34. We will determine if a 2-month course of TRAM-34, a selective inhibitor of KCa3.1, administered to APP-PS1 mice will reduce neuroinflammation, alleviate microglial dysfunction, and improve the neuropathological and behavioral outcomes of APP-PS1 mice. Because KCa3.1 blockade is relatively safe, our preclinical studies will have translational significance for future developmen of drugs for treating individuals with mild cognitive impairment or AD through inhibition of detrimental microglia functions.